ABSTRACT Congenital malformations, or structural birth defects, are now the leading cause of infant mortality in the US and Europe. Of the congenital malformations, congenital heart disease (CHD) is the most common. Mutations in the T-box transcription factor TBX5 have been found to be causative to a range of human cardiac abnormalities including Tetrology of Fallot and Holt Oram Syndrome (HOS), disease states associated associated with cardiac septal defects. While TBX5 is an essential transcription factor for heart development and its disease relevance is well established, there are many critical questions unanswered about the mechanism of how TBX5 functions. We do not understand what proteins complex with TBX5 during different stages of cardiac development and homeostasis, how these interactions regulate TBX5's choice of distinct transcriptional targets at different times, or how these interactions function to activate and/or repress target gene transcription. To this end, our labs recently initiated a directed proteomic-based approach to identify proteins that function in association with TBX5. These studies demonstrate TBX5 interacts with the transcriptional repression machinery of the Nucleosome Remodeling and Deacetylase (NuRD) complex. We further demonstrated that TBX5 human disease mutations disrupt this interaction, leading to ectopic expression of non-cardiac genes normally repressed by TBX5 and septal defects associated with Holt Oram syndrome. Collectively, this work led to the central hypothesis that TBX5 function and thus its suites of target genes are regulated during cardiac development through changes in the components of the TBX5 interactome. To address this hypothesis, we will used an integrated systems based approach to determine the mechanisms by which Tbx5 regulates distinct gene programs in the heart by defining the endogenous cardiac TBX5 transcriptional complexes, establish the mechanisms of TBX5 repression and activation and by determining the potential role of co-factors in TBX5 transcriptional regulation.